Various inflammatory and proliferative processes have been shown to be mediated through intracellular second messenger signaling associated with the activity of the Phospholipase D (PLD) enzyme.
PLD causes the hydrolysis of cell membrane phospholipids, such as the hydrolysis of phosphatidylcholine (PC), to phosphatidic acid (PA) and free choline. The hydrolysis of PC by PLD has been implicated in a variety of signal transduction pathways (Billah, M. M., (1993) Curr. Opin. Immunol. 5: 114-123; Exton, J. H., (1994) Biochem. Biophys. Acta 1212: 26-42). Phosphatidic acid (PA) has been implicated as a second messenger molecule which elicits biological responses such as enzyme release (Kanaho, et al., (1991) J. Immunol. 144: 1901-1908), the activation of protein kinase C (Stasek, Jr., et al., (1993) Biochem. Biophys. Res. Comm. 191: 134-141), activation of phospholipase C-.gamma. (Jones and Carpenter, (1993) J. Biol. Chem. 268: 20845-20850), and cellular influx of calcium (Putney, et al., (1980) Nature 284: 345-347).
PLD has also been shown to stimulate the endogenous release of PA leading to increased insulin release from islet cells (Metz and Dunlop, (1990) Biochem. J. 270: 427-435) and aldosterone secretion from the adrenal glomerulosa cells (Bollag, et al., (1990) Endocrinology 127: 1436-1443). An endogenous choline pool for the biosynthesis of acetylcholine is created by the PLD mediated cleavage of choline from PC (Chalifour and Kanfer, (1980) Biophys. Biochem. Res. Comm. 124: 945-949).
Receptor-mediated activation of PLD occurs in cells treated with cytokines, growth factors, hormones, and neurotransmitters (Natarajan & Iwamoto, (1994) Biochem. Biophys. Acta 1213: 14-20, Zhou, et al., (1993) Biochem. Pharmacol. 46: 139-148). Many of these responses are dependent on trimeric guanyl nucleotide regulatory proteins (G proteins) (Cockcroft, S., (1992) Biochem. Biophys. Acta 1113: 135-160). PLD can be activated by tyrosine phosphorylation (Dubyak, et al., (1993) Biochem. J. 292: 121-128, Gomez-Cambronero, J., (1995) J. Interferon Cytokine Res. 15: 877-885), ceramides (Gomez-Munoz, et al., (1994) J. Biol. Chem. 269: 8937-8943), and by ras superfamily GTP binding proteins (Cockcroft, et al., (1994) Science 263: 523-526, Kuribara, et al., (1995) J. Biol. Chem. 270: 25667-25671, Lambeth, et al., (1995) J. Biol. Chem. 270: 2431-2434, Massenburg, et al., (1994) Proc. Natl. Acad. Sci. U.S.A. 91: 11718-11722).
In addition to producing the lipid messenger PA, PLD activity leads to the formation of diacylglycerol (DAG), the endogenous activator of PKC, through dephosphorylation of PA by the action of PA phosphohydrolase (Kanoh, et al., (1992) J. Biol. Chem. 267: 25309-25314). The release of DAG and associated activation of PKC in leukocytes can also lead to cell proliferation and inflammatory processes (Pfeffer, et al., (1990) Proc. Natl. Acad. Sci. U.S.A., 87: 6537-6541).
Mitogenic activity associated with PA and its metabolites has been linked to the actions of the arachidonic acid derivatives which are created from further metabolism of PA (Wilkes, et al., (1993) Fed. Eur. Biochem. Soc. (FEBS) 322: 147-150; Boarder, M. R. (1994) Trends Pharmacol. Sci. 15: 57-62), These arachidonic acid derivatives of PA have been shown to inhibit the conversion of the activated GTP-bound form of ras proteins to the inactivated form which is bound to GDP (Tsai, et al., (1989) Science, 243: 522-426). Recently studies have shown that an association with increased PLD activity and multidrug resistance in breast cancer cells (Welsh, et al., (1994) Biochem. Biophys. Res. Comm., 202: 211-217). PA and its metabolite, lysophosphatidic acid, have been shown to have growth factor-like mitogenic activity in fibroblast cultures (Durieux and Lynch, (1993) Trends Pharmacol. Sci. 14: 249-254). Thus the administration of PLD inhibitors would appear to offer a viable treatment for tumours and their proliferation.
Increased intracellular PA concentrations are manifested in a diversity of cellular changes in cell cycle control (McPhail, et al., (1993) Eur. J. Haematol. 51: 294-300; Stutchfield, et al., (1993) Biochem. J. 293: 649-655; Yasui, et al., (1994) J. Immunol. 152: 5922-5929), stimulation of DNA synthesis (Fukami and Takenawa, (1992) J. Biol. Chem. 267: 10988-10993), and stimulation of c-fos and c-myc transcription (Kanuss, et al., (1990) J. Biol. Chem. 269: 12228-12233).
Additionally, PLD signaling has been found to have a stimulatory effect on actin filamentation (Ha, et al., (1994) J. Cell. Biol. 123: 1789-1796). Actin rearrangements involve severing of actin filaments, formation of nucleation sites and subsequent re-polymerization. Such events are important for cellular activities such as mobility, proliferation, and secretion. Receptor-mediated activation of PLD in whole cell experiments has also been implicated in contraction (Ohanian, et al., (1990) J. Biol. Chem. 265: 8921-8928) and phagocytosis (Fallman, et al., (1992) J. Biol. Chem. 267: 2656-2663.
The release of PA has been linked to chemotaxis, degranulation and the generation of oxygen radicals in neutrophils and other inflammatory cells (I. M. Goldstein, Complement: Biologically Active Products. In Inflammation: Basic Principles and Clinical Correlates, 55 (J. I. Gallin, I. M. Goldstein, and R. Snyderman, eds., Raven Press, N.Y., N.Y., 1988). Chemoattractants such as C5a, N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) and leukotriene B.sub.4 (LTB.sub.4) bind to cell surface receptors to initiate intracellular events such as hydrolysis of membrane phospholipids by phospholipase C, phospholipase A.sub.2, and PLD (Cockcroft, S. (1992) Biochem. Biophys. Act 11:13 135-160). Receptor stimulation in human polymorphonuclear neutrophils (PMN's), with C5a or fMet-Leu-Phe, has been shown to cause an increase in DAG predominantly as a result of PA dephosphorylation mediated by PLD (Billah, et al., (1989a) J. Biol. Chem. 264: 17069-17077; Mullmann, et al., (1990a) J. Immunol. 144: 1901-1908).
In the presence of ethanol and other short-chain alcohols, PLD catalyzes a transphosphatidylation reaction which results in the PA moiety from a phospholipid being transferred to the alcohol to produce phosphatidylethanol (PEt) (Kanfer, J. N., (1985) Can. J. Biochem. 58: 1370-1380). The formation of PEt from this reaction is widely utilized as a specific indicator of PLD activity in intact cells.
The addition of ethanol or butanol to intact cells has been shown to decrease PA production with a corresponding inhibition of secretion from mast cells (Gruchalla, et al., J. Immunol. (1990) 144: 2334-2342), platelets (Benistani and Rubin, (1990) Biochem. J. 269: 489-497), neutrophils (Yuli, et al., (1982) Proc. Natl. Acad. Sci. U.S.A. 79: 5906-5910), and differentiated HL60 cells.
Due to the lack of specific PLD inhibitors, the role of PLD activation in physiological processes, such as secretion and superoxide generation, has primarily been assessed by including primary alcohols such as ethanol or butanol into an in vitro assay to "trap" PLD generated product. In the presence of these alcohols, inhibition of free PA production is accompanied by a reduction of granule secretion or respiratory burst (Bauldry, et al., (1991) J. Biol. Chem. 266: 4173-4179); Bonser, et al., (1989) Biochem. J. 264: 617-620; Xie, et al., (1991) J. Clin. Invest. 88: 45-54; Stutchfield and Cockroft, (1993) Biochem. J. 293: 649-655; Zhou, et al., (1993) Biochem. Pharmacol. 46: 139-148). Recently, a ketoepoxide has been described which inhibited both PLD activation and superoxide generation induced by fMet-Leu-Phe in HL-60 granulocytes (Pai, et al., (1994) Anti-Cancer Drug Design 9: 363-372) lending further support for a role of PLD in the respiratory burst. Additionally, non-specific Inhibitors of PLD have been shown to decrease both fMet-Leu-Phe-induced superoxide production in HL-60 cells and platelet-derived growth factor-induced cellular growth in human fibrosarcoma cells (Pai, et al., (1994) Anti-Cancer Drug Design 9: 363-372).
Consistent with its critical role in second-messenger signaling, PLD is regulated by several mechanisms including protein phosphorylation (Dubyak et al., (1993) Biochem. J. 292:121-128), receptor-coupled G proteins (Cockroft, (1992) Biochem Biophys. Acta 1113:135-160), and small GTP binding proteins of the ras superfamily (Jiang et al., (1995) Nature 378:409-412; Lambeth et al., (1995) J. Biol. Chem. 270:2431-2434). Low molecular weight GTP-binding proteins of both the ADP-ribosylation factor (ARF) and Rho families have been shown to be required for maximal PLD activity. In addition to these regulatory components, it has been demonstrated that PLD activity is stimulated by gelsolin, a critical regulator of actin filamentation in a manner that is consistent with many observations regarding PLD signaling, inositol cycling, Ca.sup.2+ influx, and cytoskeletal reorganizations (Steed et al., (1996) Biochemistry 35:5229-5237). Since Rho proteins have been shown to play a role in both PLD (Bowman et al., 1993) and cytoskeletal regulation (Leffers et al., (1993) Experimen. Cell Res. 209:165-174), this class of proteins is likely to be involved in the PLD/gelsolin interaction. A recent report indicates that PLD activation correlates with ARF translocation to the membrane, suggesting that ARF localizes PLD to the membrane (Houle et al., (1995) J. Biol. Chem. 270:22795-22800.
Despite the intensive study dedicated to PLD and its regulatory importance, the purification of a mammalian PLD to homogeneity and the cloning of human PLD have only been reported recently (Hammond et al., 1995, Okamura & Yamashita, 1994). The partial characterization of a second isoform of PLD, PLD2, has recently been reported (Colley et al., (1997) Current Biol. 7:191-201) for mice and rat (Tsutomu and Yamashita, (1997) J. Biol. Chem. 272(17):11406-11413). Mouse PLD2 affects the regulation of the cytoskeleton, is highly enriched in brain, is localized to the cell membrane, is negatively regulated, and has high constitutive activity. This is completely consistent with all of the characteristics of a PLD from rabbit brain (Steed et al., (1996) Biochemistry 35:5229-5237; Tsutomu and Yamashita, (1997) J. Biol. Chem. 272(17):11406-11413); therefore suggesting that rabbit brain PLD is possibly PLD2. Until now, the existence and identity of a PLD2 in humans has not been known. Surprisingly, a human PLD2 has been found and its amino acid sequence determined. Human PLD2 is the subject of the present invention.